Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

Abstract

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Long Chain Fatty Acids and Virulence Repression in Intestinal Bacterial Pathogens

“…PrfA is the master virulence regulator that activates genes required for intracellular replication (Scortti et al, 2007). PrfA is comprised of a Cterminal domain that contains the HTH DNA-binding motif and an N-terminal domain that contains the dimerization domain and a ligand binding pocket (Scortti et al, 2007;Tran et al, 2022). PrfA binds its target DNA sequences called PrfA boxes as a homodimer in a hierarchical fashion (Sheehan et al, 1995).…”

“…The precise mechanism by which LCUFAs interact with PrfA to modulate its DNA binding activity remains to be elucidated (Figure 1C). Notably, small molecule inhibitors have been identified that induce a conformational shift that precludes PrfA binding to DNA (Good et al, 2016;Kuleıń et al, 2018;Tran et al, 2022). Thus, LCUFAs may also act through a similar mechanism.…”

Antibiofilm mechanism of peppermint essential oil to avert biofilm developed by foodborne and food spoilage pathogens on food contact surfaces

“…In V. parahaemolyticus (vp0952, vp0962), flaA and flgL are responsible for biofilm formation, aphA is responsible for flagella movement, and luxS is responsible for quorum sensing (Roy et al., 2022). In L. monocytogenes , PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche (Tran et al., 2022), and Listeriolysin O is a major virulence factor in this bacterium, which is encoded by hlyA (Kargar &, Ghasemi). Flagella of L. monocytogenes is composed of a basal body, hook/junction proteins, a flagellar motor/switch, a flagella export apparatus, and a flagellar filament containing mostly the flagellin protein FlaA (Dussurget, 2008).…”

Iron regulates contrasting toxicity of uropathogenic Eschericia coli in macrophages and epithelial cells

“…From the electron microscopy micrographs, we also measured the distance between the outer membrane of the bacteria and the (inner or limiting) membrane of the vacuoles at 1 hpi (Figure 4B), because the presence of a large space around bacteria in vacuoles is suggestive of proliferating bacteria [62][63][64]. The distances between the phagosomal membrane and the bacterial outer membranes were 96 nm (± 16.6; n = 32), 115 nm (± 46; n = 10), and 109 nm (± 15; n = 35) for ST999, ST131 and ST1981, respectively.…”